Chemokines constitute a family of small cytokines that are produced in inflammation and regulate leukocyte recruitment (Baggiolini, M. et al., Adv. Immunol. 55: 97-179 (1994); Springer, T. A., Annu. Rev. Physiol. 57: 827-872 (1995); and Schall, T. J. and K. B. Bacon, Curr. Opin. Immunol. 6: 865-873 (1994)). Chemokines are capable of selectively inducing chemotaxis of the formed elements of the blood (other than red blood cells), including leukocytes such as neutrophils, monocytes, macrophages, eosinophils, basophils, mast cells, and lymphocytes, including T cells and B cells. In addition to stimulating chemotaxis, other changes can be selectively induced by chemokines in responsive cells, including changes in cell shape, transient rises in the concentration of intracellular free calcium ions (Ca2+), granule exocytosis, integrin upregulation, formation of bioactive lipids (e.g., leukotrienes) and respiratory burst, associated with leukocyte activation. Thus, the chemokines are early triggers of the inflammatory response, causing inflammatory mediator release, chemotaxis and extravasation to sites of infection or inflammation.
Two subfamilies of chemokines, designated as CXC and CC chemokines, are distinguished by the arrangement of the first two of four conserved cysteine residues, which are either separated by one amino acid (as in CXC chemokines SDF-1, IL-8, IP-10, MIG, PF4, ENA-78, GCP-2, GROα, GROβ, GROγ, NAP-2, NAP-4) or are adjacent residues (as in CC chemokines MIP-1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309). Most CXC chemokines attract neutrophil leukocytes. For example, the CXC chemokines interleukin 8 (IL-8), platelet factor 4 (PF4), and neutrophil-activating peptide 2 (NAP-2) are potent chemoattractants and activators of neutrophils. The CXC chemokines designated MIG (monokine induced by gamma interferon) and IP-10 (interferon-γ inducible 10 kDa protein) are particularly active in inducing chemotaxis of activated peripheral blood lymphocytes. CC chemokines are generally less selective and can attract a variety of leukocyte cell types, including monocytes, eosinophils, basophils, T lymphocytes and natural killer cells. CC chemokines such as human monocyte chemotactic proteins 1-3 (MCP-1, MCP-2 and MCP-3), RANTES (Regulated on Activation, Normal T Expressed and Secreted), and the macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) have been characterized as chemoattractants and activators of monocytes or lymphocytes, but do not appear to be chemoattractants for neutrophils.
CC and CXC chemokines act through receptors that belong to a superfamily of seven transmembrane spanning G protein-coupled receptors (Murphy, P. M., Pharmacol Rev. 52:145-176 (2000)). This family of G-protein coupled receptors comprises a large group of integral membrane proteins, containing seven transmembrane-spanning regions. The receptors are coupled to G proteins, which are heterotrimeric regulatory proteins capable of binding GTP and mediating signal transduction from coupled receptors, for example, by the production of intracellular mediators.
Generally speaking, chemokine and chemokine receptor interactions tend to be promiscuous in that one chemokine can bind many chemokine receptors and conversely a single chemokine receptor can interact with several chemokines. There are a few exceptions to this rule; one such exception has been the interaction between SDF-1 and CXCR4 (Bleul et al., J Exp Med, 184(3): 1101-9 (1996); Oberlin et al., Nature, 382(6594): 833-5 (1996)). Originally identified as a pre-B cell growth-stimulating factor (Nagasawa et al., Proc Natl Acad Sci USA, 91(6): 2305-9 (1994)), SDF-1 has been the only reported human ligand for CXCR4. The SDF-1 gene encodes two proteins, designated SDF-1α and SDF-1β, by alternative splicing. These two proteins are identical except for the four amino acid residues that are present in the carboxy-terminus of SDF-1β and absent from SDF-1α.
The SDF-1/CXCR4 chemokine/chemokine receptor pair has been implicated as an important mediator of normal embryonic development as well as several disease states. Gene knockout data for both CXCR4 and SDF-1 demonstrate that the deletion of either this receptor or ligand proves to be embryonic lethal (Loetscher et al., J Biol Chem, 269(1): 232-7 (1994); Ma et al., Proc Natl Acad Sci USA, 95(16): 9448-53 (1998); Zou et al., Nature, 393(6685): 595-9 (1998)). Additionally, this ligand/receptor pair plays a role in HIV infection (Bleul et al., Nature, 382(6594): 829-33 (1996); Deng et al., Nature, 381(6584): 661-6 (1996); Feng et al., Science, 272(5263): 872-7 (1996)), and has been linked to HIV pathogenesis. CXCR4 can induce apoptosis and this pathway can be inhibited by SDF-1 (Berndt et al., Proc Natl Acad Sci USA, 95(21): 12556-61 (1998)), thus further supporting the evidence that SDF-1 and CXCR4 are a closely linked chemokine/chemokine receptor pair.
Most recently, SDF-1 and CXCR4 have been implicated as potentially playing a role in breast cancer metastasis (Muller et al., Nature, 410(6824): 50-6 (2001)). CXCR4 expression was detected on breast tumor cells, while elevated levels of SDF-1 were identified in the organs of metastatic growth such as lymph node, lung, liver and bone marrow, but not in other organs where tumors were not detected. Furthermore, this metastatic growth could be inhibited by the addition of anti-CXCR4, supporting the hypothesis that this ligand receptor pair has a role in tumor metastasis.
The CCR1 gene encodes a member of the beta or CC chemokine receptor family comprising 354 amino acids with predicted seven transmembrane topology (Neote, K et al. Cell 72:415-425 (1993)). The ligands of this receptor include MIP-1α, RANTES, MCP-3 and MPIF-1. CCR1 receptors are widely expressed on human leukocytes, can mediate G protein signal transduction, and are critical for the recruitment of effector immune cells to sites of inflammation and infection. Human CCR1 protein (SEQ ID NO:2) is antigenic in mice and specific mouse-anti human CCR1 monoclonal antibodies are widely available (R&D Systems, Pharmingen). Knockout studies of the mouse homolog suggest roles of this gene in host protection from inflammatory response, regulation of susceptibility to virus and parasites as well as roles in transplant rejection and tolerance. This gene and other chemokine receptor genes, including CCR2, CCR3, CCR5 are found to form a gene cluster on human chromosome 3p21 (Murphy, P. M., Pharmacol Rev. 52:145-176 (2000)).
There are many aspects of chemokine receptor signaling and selectivity for ligands that were not previously understood. The present invention addresses these and other issues.